A two-hybrid system reveals previously uncharacterized protein-protein interactions within the Helicobacter pylori
Stéphane L Benoit1,2, Stephanie Agudelo1, Robert J Maier3,4
1Department of Microbiology, The University of Georgia, 30602, Athens, Georgia.
Abstract:
Iron-sulfur (Fe-S) proteins play essential roles in all living organisms. The gastric pathogen Helicobacter pylori relies exclusively on the NIF system for biosynthesis and delivery of Fe-S clusters. Previously characterized components include two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein), and a dispensable Fe-S carrier, Nfu. Among 38 proteins previously predicted to coordinate Fe-S clusters, two proteins, HP0207 (a member of the Nbp35/ApbC ATPase family) and HP0277 (previously annotated as FdxA, a member of the YfhL ferredoxin-like family) were further studied, using a bacterial two-hybrid system approach to identify protein-protein interactions. ApbC was found to interact with 30 proteins, including itself, NifS, NifU, Nfu and FdxA, and alteration of the conserved ATPase motif in ApbC resulted in a significant (50%) decrease in the number of protein interactions, suggesting the ATpase activity is needed for some ApbC-target protein interactions. FdxA was shown to interact with 21 proteins, including itself, NifS, ApbC and Nfu, however no interactions between NifU and FdxA were detected. By use of cross-linking studies, a 51-kDa ApbC-Nfu heterodimer complex was identified. Attempts to generate apbC chromosomal deletion mutants in H. pylori were unsuccessful, therefore indirectly suggesting the hp0207 gene is essential. In contrast, mutants in the fdxA gene were obtained, albeit only in one parental strain (26695). Taken together, these results suggest both ApbC and FdxA are important players in the H. pylori NIF maturation system.
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